Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-08-09
2002-06-25
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S320100, C435S235100, C435S069100, C435S006120, C435S325000, C435S348000, C536S023100, C536S023200, C536S023400, C536S023720, C536S024100, C536S024300
Reexamination Certificate
active
06410221
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of recombinantly-produced proteins and specifically to novel, recombinant Entomopoxvirus genes, proteins, protein regulatory sequences and their uses in expressing heterologous genes in transformed hosts.
BACKGROUND OF THE INVENTION
Poxviruses are taxonomically classified into the family Chordopoxvirinae, whose members infect vertebrate hosts, e.g., the Orthopoxvirus vaccinia, or into the family Entomopoxvirinae. Very little is known about members of the Entomopoxvirinae family other than the insect host range of individual members. One species of Entomopoxvirus (EPV) is the
Amsacta moorei
Entomopoxvirus (AmEPV), which was first isolated from larvae of the red hairy caterpillar
Amsacta moorei
(Roberts and Granados [1968
] J. Invertebr. Pathol
. 12:141-143). AmEPV is the type species of genus B of EPVs and is one of three known EPVs which will replicate in cultured insect cells (R. R. Granados et al. [1976] “Replication of
Amsacta moorei
Entomopoxvirus and
Autographa californica
Nuclear Polyhedrosis Virus in Hemocyte Cell Lines from
Estigmene acrea
,” in
Invertebrate Tissue Culture Applications in Medicine, Biology, and Agriculture
, E. Kurstak and K. Maramorosch (ed.), Academic Press, New York, pp. 379-389; T. Hukuhara et al. [1990
] J. Invertebr. Pathol
. 56:222-232; and Sato, T. [1989] “Establishment of Eight Cell Lines from Neonate Larvae of Torticids (Lepidoptera), and Their Several Characteristics Including Susceptibility to Insect Viruses,” in
Invertebrate Cell Systems Applications
, J. Mitsuhashi (ed.), Vol. II, CRC Press, Inc., Boca Raton, Fla., pp. 187-198).
AmEPV is one of the few insect poxviruses which can replicate in insect cell culture; AmEPV is unable to replicate in vertebrate cell lines. The AmEPV double-stranded DNA genome is about 225 kb and is unusually A+T rich (18.5% G+C) (W. Langridge, H. R., et al. [1977
] Virology
76:616-620). Recently, a series of restriction maps for AmEPV were published (Hall, R. L., et al. [1990
] Arch. Virol
. 110:77-90). No DNA homology to vaccinia has been detected (Langridge, W. H. [1983
] J. Invertebr. Pathol
. 42:77-82; Langridge, W. H. [1984
] J. Invertebr. Pathol
. 43:41-46).
The viral replication cycle of AmEPV resembles that of other poxviruses except for the appearance of occluded virus late in infection. For AmEPV, once a cell is infected, both occluded and extracellular virus particles are generated. The mature occlusion body particle, which is responsible for environmentally protecting the virion during infection, consists of virus embedded within a crystalline matrix consisting primarily of a single protein, spheroidin. Spheroidin, the major structural protein of AmEPV, has been reported to be 110 kDa in molecular weight and to consist of a high percentage of charged and sulfur-containing amino acids (Langridge and Roberts [1982
] J. Invertebr. Pathol
. 39:346-353).
Another insect virus is the baculovirus. Like baculoviruses, a characteristic feature of entomopoxviruses is the amalgamation of virions within environmentally stable occlusion bodies. It is this occluded form of the virus that is primarily responsible for dissemination to other insects. While the major protein (polyhedrin) of baculovirus occlusions is quite similar between viruses, it has been reported that the major occlusion body protein (spheroidin) of two group B entomopoxviruses,
Amsacta moorei
(AmEPV) and
Choristoneura biennis
(CbEPV) is quite different both in terms of amino acid sequence and coding capacity of the corresponding spheroidin genes (115 and 47 kDa for AmEPV and CbEPV, respectively).
The entomopoxviruses and the role of occlusion bodies have recently been reviewed by Arif and Kurstak (Arif, B. M., E. Kurstak, E. [1991 ] “The entomopoxviruses,” In
Viruses of Invertebrates
(E. Kurstak, Ed.), pp. 179-195, Marcel Dekker, Inc., New York) and Goodwin et al. (Goodwin, R. H., R. J. Milner, C. D. Beaton [1991] “Entomopoxvirinae,” In
Atlas of Invertebrate Viruses
(J. R. Adams and J. R. Bonami, Eds.), pp. 259-285, CRC Press, Inc., Boca Raton). The gene which encodes the AmEPV spheroidin, a 115 kDa protein, has been identified and sequenced (Hall, R. L., R. W. Moyer [1991
] J. Virol
. 65, 6516-6527; Banville, M., F. Dumas, F., S. Trifiro, B. Arif, C. Richardson [1992] J. Gen. Virol. 73, 559-566). The AmEPV gene was also mapped and found to be located at the 3′ end of a nucleoside triphosphate phosphohydrolase gene (NPH I or NTPase I, Hall and Moyer [1991], supra). The spheroidin gene of
Choristoneura biennis
entomopoxvirus (CbEPV) has been reported to be derived from a gene capable of encoding a 47 kDa protein (Yuen, L., J. Dionne, B. Arif, C. Richardson [1990
] Virology
175:427-433.). A comparison of the sequence of the two spheroidins shows no relationship between the two encoded proteins.
We have investigated the spheroidin genes of
Choristoneura biennis, Choristoneura fumiferana
, and
Amsacta moorei
viruses. Our results indicate, in contrast to published results, that the initial Choristoneura EPV spheroidin assignment is likely incorrect and that the Choristoneura spheroidin is instead a highly conserved homolog of the AmEPV spheroidin.
The use of viruses and virus proteins in eukaryotic host-vector systems has been the subject of a considerable amount of investigation and speculation. Many existing viral vector systems suffer from significant disadvantages and limitations which diminish their utility. For example, a number of eukaryotic viral vectors are either tumorigenic or oncogenic in mammalian systems, creating the potential for serious health and safety problems associated with resultant gene products and accidental infections. Further, in some eukaryotic host-viral vector systems, the gene product itself exhibits antiviral activity, thereby decreasing the yield of that protein.
In the case of simple viruses, the amount of exogenous DNA which can be packaged into a simple virus is limited. This limitation becomes a particularly acute problem when the genes used are eukaryotic. Because eukaryotic genes usually contain intervening sequences, they are too large to fit into simple viruses. Further, because they have many restriction sites, it is more difficult to insert exogenous DNA into complex viruses at specific locations.
Vaccinia virus has recently been developed as an eukaryotic cloning and expression vector (Mackett, M., et al. [1985
] DNA Cloning
, Vol. II, ed. D. M. Glover, Oxford: IRL Press, pp. 191-212; Panicali, D., et al. [1982
] Proc. Natl. Acad. Sci. USA
, 88:5364-5368). Numerous viral antigens have been expressed using vaccinia virus vectors (Paoletti, E., et al. [1984
] Proc. Natl. Acad. Sci. USA
81:193-197; Piccine, A., et al. [1986
] BioEssays
5:248-252) including, among others, HBsAg, rabies G protein and the gp120/gp41 of human immunodeficiency virus (HIV). Regulatory sequences from the spruce budworm EPV have been used previously with vaccinia (Yuen, L., et al. [1990
] Virology
175:427-433).
Additionally, studies with vaccinia virus have demonstrated that poxviruses have several advantageous features as vaccine vectors. These include the ability of poxvirus-based vaccines to stimulate both cell-mediated and humoral immunity, minimal cost to mass produce vaccine and the stability of the lyophilized vaccine without refrigeration, ease of administration under non-sterile conditions, and the ability to insert at least 25,000 base pairs of foreign DNA into an infectious recombinant, thereby permitting the simultaneous expression of many antigens from one recombinant.
There exists a need in the art for additional viral compositions and methods for use in expressing heterologous genes in selected host cells, and in performing other research and production techniques associated therewith. In addition, it is noted
Gruidl Michael E.
Hall Richard L.
Li Yi
Moyer Richard W.
Guzo David
Saliwanchik Lloyd & Saliwanchik
University of Florida Research Foundation Inc.
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